Packing materials for liquid chromatography (LC) are generally classified into two types: those having organic or polymeric carriers, e.g., polystyrene polymers; and those having inorganic carriers typified by silica gel. The polymeric materials are chemically stable against alkaline and acidic mobile phases; therefore, the pH range of the eluent used with polymeric chromatographic materials is wide, compared with the silica carriers. However, polymeric chromatographic materials generally result in columns having low efficiency, leading to inadequate separation performance, particularly with low molecular-weight analytes. Furthermore, polymeric chromatographic materials shrink and swell upon solvent changeover in the eluting solution.
On the other hand, silica gel-based chromatographic devices, e.g., HPLC columns, are most commonly used. The most common applications employ a silica which has been surface-derivatized with an organic functional group such as octadecyl (C18), octyl (C8), phenyl, amino, cyano group, etc. As a stationary phase for HPLC, these packing materials result in columns with high theoretical plate number/high efficiency, and do not evidence shrinking or swelling. Silica gel is characterized by the presence of silanol groups on its surface. During a typical derivatization process such as reaction with octadecyldimethylchlorosilane, at least 50% of the surface silanol groups remain unreacted.
A drawback with silica-based columns is their limited hydrolytic stability. First, the incomplete derivatization of the silica gel leaves a bare silica surface which can be readily dissolved under alkaline conditions, generally pH greater than 8.0, leading to the subsequent collapse of the chromatographic bed. Secondly, the bonded phase can be stripped off of the surface under acidic conditions, generally pH less than 2.0, and eluted off the column by the mobile phase, causing loss of analyte retention, and an increase in the concentration of surface silanol groups. These problems have been attributed to free silanol group activity and hydrolytic instability of silica-based stationary phases. To address to these problem, many methods have been tried including use of ultrapure silica, carbonized silica, coating of the silica surface with polymeric materials, endcapping free silanol groups with a short-chain reagent such as trimethylchlorosilane, and the addition of suppressors such as amines to the eluent. These approaches have not proven to be completely satisfactory in practice.
Hybrid columns which combine organic and silica systems are known (XTerra(trademark) MS C18 (Waters Corp., Milford, Mass. USA) and offer, potentially, the benefits of both silica and organic based materials. Hybrid particles have the advantages of both silica and polymer packing materials. In particular, hybrid particles offer mechanical strength, high efficiency, ability to separate a wide range of compounds, high chemical and temperature stability with little to no peak tailing, and improved peak shape for basic compounds. However, these materials have certain limitations, also.
Many of the limitations of hybrid silica-based columns can be attributed to surface organic (i.e., methyl groups). In particular, the presence of surface organic groups lead to lower bonded phase surface concentrations after bonding with silanes, e.g., C18 and C8 silanes, in comparison to silica phases, presumably because the methyl groups on the surface are unreactive to bonding. Further, in C18 bonded phases, surface organic groups may decrease the level of cross-bonding between adjacent C18 ligands. This results in reduced low pH stability since the C18 ligand has fewer covalent bonds to the surface of the particle. Ultimately, reduced retention times and peak compression can result from the reduced low pH stability caused by surface organic groups.
The present invention relates to improved hybrid chromatographic materials which demonstrate improved stability and separation characteristics. The chromatographic hybrid particles can be used for performing separations or for participating in chemical reactions. These particles feature a surface with a desired bonded phase, e.g., ODS or CN, and a controlled surface concentration of silicon-methyl groups. More particularly, surface silicon-methyl groups are selectively replaced with silanol groups. In so doing, the hybrid particles have substantially improved low pH stability, and improved chromatographic separation performance including reduced peak tailing.
In an embodiment, particles of the invention have an interior area and an exterior surface and are of a composition represented by:
[A]y[B]xxe2x80x83xe2x80x83(Formula I)
where x and y are whole number integers and A is represented by:
SiO2/(R1pR2qSiOt)nxe2x80x83xe2x80x83(Formula II),
and/or
SiO2/[R3(R1rSiOt)m]nxe2x80x83xe2x80x83(Formula III);
where R1 and R2 are independently a substituted or unsubstituted C1 to C7 alkyl group or a substituted or unsubstituted aryl group, R3 is a substituted or unsubstituted C1 to C7 alkylene, alkenylene, alkynylene, or arylene group bridging two or more silicon atoms, p and q are 0, 1, or 2, provided that p+q=1 or 2, and that when p+q=1, t=1.5, and when p+q=2, t=1; r is 0 or 1, provided that when r=0, t=1.5, and when r=1, t=1; m is an integer greater than or equal to 2; and n is a number from 0.01 to 100. B is represented by:
SiO2/(R4vSiOt)nxe2x80x83xe2x80x83(Formula IV)
where R4 may be hydroxyl, fluorine, alkoxy (e.g., methoxy), aryloxy, substituted siloxane, protein, peptide, carbohydrate, nucleic acid, and combinations thereof, and R4 is not R1, R2, or R3. v is 1 or 2, provided that when v=1, t=1.5, and when v=2, t=1; and n is a number from 0.01 to 100. The interior of the particle has a composition of A, the exterior surface of the particle has a composition represented by A and B, and the exterior composition is between about 1 and about 99% of the composition of B and the remainder including A. In these particles, R4 may be represented by:
xe2x80x94OSi(R5)2xe2x80x94R6xe2x80x83xe2x80x83(Formula V)
where R5 may be a C1 to C6 straight, cyclic, or branched alkyl, aryl, or alkoxy group, a hydroxyl group, or a siloxane group, and R6 may be a C1 to C36 straight, cyclic, or branched alkyl (e.g., C18, cyanopropyl), aryl, or alkoxy group, where the groups of R6 are unsubstituted or substituted with one or more moieties such as halogen, cyano, amino, diol, nitro, ether, carbonyl, epoxide, sulfonyl, cation exchanger, anion exchanger, carbamate, amide, urea, peptide, protein, carbohydrate, and nucleic acid functionalities.
In another embodiment, R6 may greater than about 2.5 xcexcmol/m2, more preferably greater than about 3.0 xcexcmol/m2, and still more preferably greater than about 3.5 xcexcmol/m2. In a preferred embodiment, the surface concentration of R6 is between about 2.5 and about 3.7 xcexcmol/m2.
This invention further provides a method of preparation of particles for performing separations or for participating in chemical reactions, including: prepolymerizing a mixture of an organoalkoxysilane and a tetraalkoxysilane (e.g., tetramethoxysilane and tetraethoxysilane) in the presence of an acid catalyst to produce a polyalkoxysiloxane; preparing an aqueous surfactant containing suspension of the polyalkoxysiloxane, and gelling in the presence of a base catalyst so as to produce porous particles having silicon C1 to C7 alkyl groups, substituted or unsubstituted aryl groups, substituted or unsubstituted C1 to C7 alkylene, alkenylene, alkynylene, or arylene groups; modifying the pore structure of the porous particles by hydrothermal treatment; and replacing one or more surface C1 to C7 alkyl groups, substituted or unsubstituted aryl groups, substituted or unsubstituted C1 to C7 alkylene, alkenylene, alkynylene, or arylene groups of the particle with hydroxyl, fluorine, alkoxy, aryloxy, or substituted siloxane groups.
The present invention will be more fully understood by reference to the definitions set forth below.
xe2x80x9cHybridxe2x80x9d, e.g., as in xe2x80x9cporous inorganic/organic hybrid particlesxe2x80x9d includes inorganic-based structures wherein an organic functionality is integral to both the internal or xe2x80x9cskeletalxe2x80x9d inorganic structure as well as the hybrid material surface. The inorganic portion of the hybrid material may be, e.g., alumina, silica, titanium or zirconium oxides, or ceramic material; in a preferred embodiment, the inorganic portion is silica. As noted before, exemplary hybrid materials are shown in U.S. Pat. No. 4,017,528. In a preferred embodiment where the inorganic portion is silica, xe2x80x9chybrid silicaxe2x80x9d refers to a material having the formula SiO2/(R1pR2qSiOt)n or SiO2/[R3(R1rSiOt)m]n; wherein R1 and R2 are independently a substituted or unsubstituted C1 to C7 alkyl group, or a substituted or unsubstituted aryl group, R3 is a substituted or unsubstituted C1 to C7 alkylene, alkenylene, alkynylene, or arylene group bridging two or more silicon atoms, p and q are 0, 1, or 2, provided that p+q=1 or 2, and that when p+q=1, t=1.5, and when p+q=2, t=1; r is 0 or 1, provided that when r=0, t=1.5, and when r=1, t=1; m is an integer greater than or equal to 2; and n is a number from 0.01 to 100.
A xe2x80x9cbonded phasexe2x80x9d can be formed by adding functional groups to the surface of hybrid silica. The surface of hybrid silica contains silanol groups, which can be reacted with a reactive organosilane to form a xe2x80x9cbonded phasexe2x80x9d. Bonding involves the reaction of silanol groups at the surface of the hybrid particles with halo or alkoxy substituted silanes, thus producing a Sixe2x80x94Oxe2x80x94Sixe2x80x94C linkage.
Generally, only a maximum of 50% of the Sixe2x80x94OH groups on heat treated silica can react with the trimethylsilyl entity, and less with larger entities such as the octadecylsilyl groups. Factors tending to increase bonding coverage include: silanizing twice, using a large excess of silanizing reagent, using a trifunctional reagent, silanizing in the presence of acid scavenger, performing secondary hydroxylation of the surface to be silanized, using a chlorinated solvent in preference to a hydrocarbon, and capping of the surface.
Some adjacent vicinal hydroxyls on the silica surface are at a distance such that difunctional reactions can occur between the silica surface and a difunctional or trifunctional reagent. When the adjacent hydroxyls on the silica surface are not suitably spaced for a difunctional reaction, then only a monofunctional reaction takes place.
Silanes for producing bonded silica include, in decreasing order of reactivity: RSiX3, R2SiX2, and R3SiX, where X is halo (e.g., chloro) or alkoxy. Specific silanes for producing bonded silica, in order of decreasing reactivity, include C8xe2x80x94N(CH3)2, n-octyldimethyl(trifluoroacetoxy)silane (C8xe2x80x94OCOCF3), n-octyldimethylchlorosilane (C8xe2x80x94Cl), n-octyldimethylmethoxysilane (C8xe2x80x94OCH3), n-octyldimethylethoxysilane (C8xe2x80x94OC2H5), and bis-(n-octyldimethylsiloxane) (C8xe2x80x94Oxe2x80x94C8). Monochlorosilane is the cheapest and most commonly used silane.
Other monochlorosilanes that can be used in producing bonded silica include: Clxe2x80x94Si(CH3)2xe2x80x94(CH2)nxe2x80x94X, where X is H, CN, fluorine, chlorine, bromine, iodine, phenyl, cyclohexyl, dimethylamine, or vinyl, and n is 1 to 30 (preferably 2 to 20, more preferably 8 to 18); Clxe2x80x94Si(CH3)2xe2x80x94(CH2)8xe2x80x94H (n-octyldimethylsilyl); Clxe2x80x94Si(CH(CH3)2)2xe2x80x94(CH2)nxe2x80x94X, where X is H, CN, fluorine, chlorine, bromine, iodine, phenyl, cyclohexyl, dimethylamine, or vinyl; and Clxe2x80x94Si(CH(Phenyl)2)2xe2x80x94(CH2)nxe2x80x94X where X is H, CN, fluorine, chlorine, bromine, iodine, phenyl, cyclohexyl, dimethylamine, or vinyl.
Dimethylmonochlorosilane (Clxe2x80x94Si(CH3)2xe2x80x94R) can be synthesized by a 2-step process such as shown below.
CnH2n+1xe2x80x94Br+Mgxe2x86x92CnH2n+1xe2x80x94MgBr
CnH2n+1MgBr+(CH3)2SiCl2xe2x86x92CnH2n+1Si(CH3)2Cl
Alternatively, dimethylmonochlorosilane (Clxe2x80x94Si(CH3)2xe2x80x94R) can be synthesized by a one-step catalytic hydrosilylation of terminal olefins. This reaction favors formation of the anti-Markovnikov addition product. The catalyst used may be hexachloroplatinic acid-hexahydrate (H2PtCl6-6H2O). 
The surface derivatization of the hybrid silica is conducted according to standard methods, for example by reaction with octadecyldimethylchlorosilane in an organic solvent under reflux conditions. An organic solvent such as toluene is typically used for this reaction. An organic base such as pyridine or imidazole is added to the reaction mixture to catalyze the reaction. The thus-obtained product is then washed with water, toluene and acetone and dried at 100xc2x0 C. under reduced pressure for 16 h.
xe2x80x9cFunctionalizing groupxe2x80x9d includes (typically) organic functional groups which impart a certain chromatographic functionality to a chromatographic stationary phase, including, e.g., octadecyl (C18), phenyl, ion exchange, etc. Such functionalizing groups are present in, e.g., surface modifiers such as disclosed herein which are attached to the base material, e.g., via derivatization or coating and later crosslinking, imparting the chemical character of the surface modifier to the base material. In an embodiment, such surface modifiers have the formula Za(R5)bSixe2x80x94R, where Z=Cl, Br, I, C1-C5 alkoxy, dialkylamino, e.g., dimethylamino or trifluoromethanesulfonate; a and b are each an integer from 0 to 3 provided that a+b=3; R5 is a C1-C6 straight, cyclic or branched alkyl group, and R is a functionalizing group. R5 may be methyl, ethyl, propyl, isopropyl, butyl, t-butyl, sec-butyl, pentyl, isopentyl, hexyl or cyclohexyl; preferably, Rxe2x80x2 is methyl.
The functionalizing group R may include alkyl, aryl, cyano, amino, diol, nitro, cation or anion exchange groups, or embedded polar functionalities. Examples of suitable R functionalizing groups include C1-C20 alkyl such as octyl (C8) and octadecyl (C18); alkaryl, e.g., C1-C4-phenyl; cyanoalkyl groups, e.g., cyanopropyl; diol groups, e.g., propyldiol; amino groups, e.g., aminopropyl; and embedded polar functionalities, e.g., carbamate functionalities such as disclosed in U.S. Pat. No. 5,374,755. In a preferred embodiment, the surface modifier may be a haloorganosilane, such as octyldimethylchlorosilane or octadecyldimethylchlorosilane. Embedded polar functionalities include carbamate functionalities such as disclosed in U.S. Pat. No. 5,374,755. Such groups include those of the general formula 
wherein 1, m, o, r, and s are 0 or 1, n is 0, 1, 2 or 3 p is 0, 1, 2, 3 or 4 and q is an integer from 0 to 19; R3 is selected from the group consisting of hydrogen, alkyl, cyano and phenyl; and Z, Rxe2x80x2, a and b are defined as above. Preferably, the carbamate functionality has the general structure indicated below: 
wherein R5 may be, e.g., cyanoalkyl, t-butyl, butyl, octyl, dodecyl, tetradecyl, octadecyl, or benzyl. Advantageously, R5 is octyl or octadecyl. In a preferred embodiment, the surface modifier may be a haloorganosilane, such as octyldimethylchlorosilane or octadecyldimethylchlorosilane. In another embodiment, the particles are surface modified by polymer coating.
A chromatographic stationary phase is said to be xe2x80x9cendcappedxe2x80x9d when a small silylating agent, such as trimethylchlorosilane, is used to bond residual silanol groups on a packing surface. It is most often used with reversed-phase packings and may cut down on undesirable adsorption of basic or ionic compounds. For example, end capping occurs when bonded hybrid silica is further reacted with a short-chain silane such as trimethylchlorosilane to endcap the remaining silanol groups. The goal of end capping is to remove as many residual silanols as possible. In order of decreasing reactivity, agents that can be used as trimethylsilyl donors for end capping include trimethylsilylimidazole (TMSIM), bis-N,O-trimethylsilyltrifluoroacetamide (BSTFA), bis-N,O-trimethylsilylacetamide (BSA), trimethylsilyldimethylamine (TMSDMA), trimethylchlorosilane (TMS), and hexamethyldisilane (HMDS). Preferred end-capping reagents include trimethylchlorosilane (TMS), trimethylchlorosilane (TMS) with pyridine, and trimethylsilylimidazole (TMSIM).
xe2x80x9cPorogensxe2x80x9d are described in Small et al., U.S. Pat. No. 6,027,643. A porogen is an added material which, when removed after the polymerization is complete, increases the porosity of a hybrid particle. The porosity should be such that it provides for a ready flow of liquids through the polymer phase while at the same time providing adequate areas of contact between the polymer and liquid phase. The porogen can be a finely divided solid which can be easily removed by dissolution in acid or base (e.g., calcium carbonate or silica), or it can be a solvent which is rejected by the polymer as it forms and is subsequently displaced by another solvent or water. Suitable liquid porogens include an alcohol, e.g., used in the manner described in Analytical Chemistry, Vol. 68, No.2, pp. 315-321, Jan. 15, 1996. Reverse micellular systems obtained by adding water and suitable surfactant to a polymerizable monomer have been described as porogens by Menger et al., J Am Chem Soc (1990) 112:1263-1264. Other examples of porogens can be founds in Li et al., U.S. Pat. No. 5,168,104 and Mikes et al., U.S. Pat. No. 4,104,209.
xe2x80x9cPorosityxe2x80x9d is the ratio of the volume of a particle""s interstices to the volume of the particle""s mass.
xe2x80x9cPore volumexe2x80x9d is the total volume of the pores in a porous packing, and is usually expressed in mL/g. It can be measured by the BET method of nitrogen adsorption or by mercury intrusion, where Hg is pumped into the pores under high pressure. As described in Quinn et al. U.S. Pat. No. 5,919,368, xe2x80x9cpore volumexe2x80x9d can be measured by injecting acetone into beds as a total permeating probe, and subsequently a solution of 6xc3x97106 molecular weight polystyrene as a totally excluded probe. The transit or elution time through the bed for each standard can be measured by ultra-violet detection at 254 nm. Percent intrusion can be calculated as the elution volume of each probe less the elution volume of the excluded probe, divided by the pore volume. Alternatively, pore volume can be determined as described in Perego et al. U.S. Pat. No. 5,888,466 by N2 adsorption/desorption cycles at 77xc2x0 K, using a Carlo Erba Sorptomatic 1900 apparatus.
As described in Chieng et al. U.S. Pat. No. 5,861,110, xe2x80x9cpore diameterxe2x80x9d can be calculated from 4V/S BET, from pore volume, or from pore surface area. The pore diameter is important because it allows free diffusion of solute molecules so they can interact with the stationary phase. 60 xc3x85 and 100 xc3x85 pore diameters are most popular. For packings used for the separation of biomolecules, pore diameters greater than 300 xc3x85 are used.
As also described by Chieng et al. in the ""110 Patent, xe2x80x9cparticle surface areaxe2x80x9d can be determined by single point or multiple point BET. For example, multipoint nitrogen sorption measurements can be made on a Micromeritics ASAP 2400 instrument. The specific surface area is then calculated using the multipoint BET method, and the average pore diameter is the most frequent diameter from the log differential pore volume distribution (dV/dlog(D) vs. D Plot). The pore volume is calculated as the single point total pore volume of pores with diameters less than ca. 3000 xc3x85.
xe2x80x9cParticle sizexe2x80x9d may be measured, e.g., using a Beckman Coulter Multisizer 3 instrument as follows. Particles are suspended homogeneously in a 5% lithium chloride methanol solution. A greater than 70,000 particle count may be run using a 30 xcexcm aperture in the volume mode for each sample. Using the Coulter principle, volumes of particles are converted to diameter, where a particle diameter is the equivalent spherical diameter, which is the diameter of a sphere whose volume is identical to that of the particle. Particle size can also be determined by light microscopy.
The term xe2x80x9caliphatic groupxe2x80x9d includes organic compounds characterized by straight or branched chains, typically having between 1 and 22 carbon atoms. Aliphatic groups include alkyl groups, alkenyl groups and alkynyl groups. In complex structures, the chains can be branched or cross-linked. Alkyl groups include saturated hydrocarbons having one or more carbon atoms, including straight-chain alkyl groups and branched-chain alkyl groups. Such hydrocarbon moieties may be substituted on one or more carbons with, for example, a halogen, a hydroxyl, a thiol, an amino, an alkoxy, an alkylcarboxy, an alkylthio, or a nitro group. Unless the number of carbons is otherwise specified, xe2x80x9clower aliphaticxe2x80x9d as used herein means an aliphatic group, as defined above (e.g., lower alkyl, lower alkenyl, lower alkynyl), but having from one to six carbon atoms. Representative of such lower aliphatic groups, e.g., lower alkyl groups, are methyl, ethyl, n-propyl, isopropyl, 2-chloropropyl, n-butyl, sec-butyl, 2-aminobutyl, isobutyl, tert-butyl, 3-thiopentyl, and the like.
As used herein, the term xe2x80x9cnitroxe2x80x9d means xe2x80x94NO2; the term xe2x80x9chalogenxe2x80x9d designates xe2x80x94F, xe2x80x94Cl, xe2x80x94Br or xe2x80x94I; the term xe2x80x9cthiolxe2x80x9d means SH; and the term xe2x80x9chydroxylxe2x80x9d means xe2x80x94OH.
The term xe2x80x9calicyclic groupxe2x80x9d includes closed ring structures of three or more carbon atoms. Alicyclic groups include cycloparaffins which are saturated cyclic hydrocarbons, cycloolefins and naphthalenes which are unsaturated with two or more double bonds, and cycloacetylenes which have a triple bond. They do not include aromatic groups. Examples of cycloparaffins include cyclopropane, cyclohexane, and cyclopentane. Examples of cycloolefins include cyclopentadiene and cyclooctatetraene. Alicyclic groups also include fused ring structures and substituted alicyclic groups such as alkyl substituted alicyclic groups. In the instance of the alicyclics such substituents can further comprise a lower alkyl, a lower alkenyl, a lower alkoxy, a lower alkylthio, a lower alkylamino, a lower alkylcarboxyl, a nitro, a hydroxyl, xe2x80x94CF3, xe2x80x94CN, or the like.
The term xe2x80x9cheterocyclic groupxe2x80x9d includes closed ring structures in which one or more of the atoms in the ring is an element other than carbon, for example, nitrogen, sulfur, or oxygen. Heterocyclic groups can be saturated or unsaturated and heterocyclic groups such as pyrrole and furan can have aromatic character. They include fused ring structures such as quinoline and isoquinoline. Other examples of heterocyclic groups include pyridine and purine. Heterocyclic groups can also be substituted at one or more constituent atoms with, for example, a halogen, a lower alkyl, a lower alkenyl, a lower alkoxy, a lower alkylthio, a lower alkylamino, a lower alkylcarboxyl, a nitro, a hydroxyl, xe2x80x94CF3, xe2x80x94CN, or the like. Suitable heteroaromatic and heteroalicyclic groups generally will have 1 to 3 separate or fused rings with 3 to about 8 members per ring and one or more N, O or S atoms, e.g. coumarinyl, quinolinyl, pyridyl, pyrazinyl, pyrimidyl, furyl, pyrrolyl, thienyl, thiazolyl, oxazolyl, imidazolyl, indolyl, benzofuranyl, benzothiazolyl, tetrahydrofuranyl, tetrahydropyranyl, piperidinyl, morpholino and pyrrolidinyl.
The term xe2x80x9caromatic groupxe2x80x9d includes unsaturated cyclic hydrocarbons containing one or more rings. Aromatic groups include 5- and 6-membered single-ring groups which may include from zero to four heteroatoms, for example, benzene, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, triazole, pyrazole, pyridine, pyrazine, pyridazine and pyrimidine, and the like. The aromatic ring may be substituted at one or more ring positions with, for example, a halogen, a lower alkyl, a lower alkenyl, a lower alkoxy, a lower alkylthio, a lower alkylamino, a lower alkylcarboxyl, a nitro, a hydroxyl, xe2x80x94CF3, xe2x80x94CN, or the like.
The term xe2x80x9calkylxe2x80x9d includes saturated aliphatic groups, including straight-chain alkyl groups, branched-chain alkyl groups, cycloalkyl (alicyclic) groups, alkyl substituted cycloalkyl groups, and cycloalkyl substituted alkyl groups. In preferred embodiments, a straight chain or branched chain alkyl has 20 or fewer carbon atoms in its backbone (e.g., C1-C20 for straight chain, C3-C20 for branched chain), and more preferably 12 or fewer. Likewise, preferred cycloalkyls have from 4-10 carbon atoms in their ring structure, and more preferably have 4-7 carbon atoms in the ring structure. The term xe2x80x9clower alkylxe2x80x9d refers to alkyl groups having from 1 to 6 carbons in the chain, and to cycloalkyls having from 3 to 6 carbons in the ring structure.
Moreover, the term xe2x80x9calkylxe2x80x9d (including xe2x80x9clower alkylxe2x80x9d) as used throughout the specification and claims includes both xe2x80x9cunsubstituted alkylsxe2x80x9d and xe2x80x9csubstituted alkylsxe2x80x9d, the latter of which refers to alkyl moieties having substituents replacing a hydrogen on one or more carbons of the hydrocarbon backbone. Such substituents can include, for example, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, cyano, amino (including alkyl amino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfate, sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl, aralkyl, or an aromatic or heteroaromatic moiety. It will be understood by those skilled in the art that the moieties substituted on the hydrocarbon chain can themselves be substituted, if appropriate. Cycloalkyls can be further substituted, e.g., with the substituents described above. An xe2x80x9caralkylxe2x80x9d moiety is an alkyl substituted with an aryl, e.g., having 1 to 3 separate or fused rings and from 6 to about 18 carbon ring atoms, (e.g., phenylmethyl (benzyl)).
The term xe2x80x9calkylaminoxe2x80x9d as used herein means an alkyl group, as defined herein, having an amino group attached thereto. Suitable alkylamino groups include groups having 1 to about 12 carbon atoms, preferably from 1 to about 6 carbon atoms. The term xe2x80x9calkylthioxe2x80x9d refers to an alkyl group, as defined above, having a sulfhydryl group attached thereto. Suitable alkylthio groups include groups having 1 to about 12 carbon atoms, preferably from 1 to about 6 carbon atoms. The term xe2x80x9calkylcarboxylxe2x80x9d as used herein means an alkyl group, as defined above, having a carboxyl group attached thereto. The term xe2x80x9calkoxyxe2x80x9d as used herein means an alkyl group, as defined above, having an oxygen atom attached thereto. Representative alkoxy groups include groups having 1 to about 12 carbon atoms, preferably 1 to about 6 carbon atoms, e.g., methoxy, ethoxy, propoxy, tert-butoxy and the like. The terms xe2x80x9calkenylxe2x80x9d and xe2x80x9calkynylxe2x80x9d refer to unsaturated aliphatic groups analogous to alkyls, but which contain at least one double or triple bond respectively. Suitable alkenyl and alkynyl groups include groups having 2 to about 12 carbon atoms, preferably from 1 to about 6 carbon atoms.
The term xe2x80x9carylxe2x80x9d includes 5- and 6-membered single-ring aromatic groups that may include from zero to four heteroatoms, for example, unsubstituted or substituted benzene, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, triazole, pyrazole, pyridine, pyrazine, pyridazine and pyrimidine, and the like. Aryl groups also include polycyclic fused aromatic groups such as naphthyl, quinolyl, indolyl, and the like. The aromatic ring can be substituted at one or more ring positions with such substituents, e.g., as described above for alkyl groups. Suitable aryl groups include unsubstituted and substituted phenyl groups. The term xe2x80x9caryloxyxe2x80x9d as used herein means an aryl group, as defined above, having an oxygen atom attached thereto. The term xe2x80x9caralkoxyxe2x80x9d as used herein means an aralkyl group, as defined above, having an oxygen atom attached thereto. Suitable aralkoxy groups have 1 to 3 separate or fused rings and from 6 to about 18 carbon ring atoms, e.g., O-benzyl.
The term xe2x80x9camino,xe2x80x9d as used herein, refers to an unsubstituted or substituted moiety of the formula xe2x80x94NRaRb, in which Ra and Rb are each independently hydrogen, alkyl, aryl, or heterocyclyl, or Ra and Rb, taken together with the nitrogen atom to which they are attached, form a cyclic moiety having from 3 to 8 atoms in the ring. Thus, the term xe2x80x9caminoxe2x80x9d includes cyclic amino moieties such as piperidinyl or pyrrolidinyl groups, unless otherwise stated. An xe2x80x9camino-substituted amino groupxe2x80x9d refers to an amino group in which at least one of Ra and Rb, is further substituted with an amino group.
This invention provides a particle for performing separations or for participating in chemical reactions, said particle having an interior area and an exterior surface, said particle having a composition represented by Formula I as set forth below:
[A]y[B]xxe2x80x83xe2x80x83(Formula I)
where x and y are whole number integers and A is represented by Formula II and/or Formula III below:
SiO2/(R1pR2qSiOt)nxe2x80x83xe2x80x83(Formula II),
and/or
SiO2/[R3(R1rSiOt)m]nxe2x80x83xe2x80x83(Formula III);
where R1 and R2 are independently a substituted or unsubstituted C1 to C7 alkyl group or a substituted or unsubstituted aryl group, R3 is a substituted or unsubstituted C1 to C7 alkylene, alkenylene, alkynylene, or arylene group bridging two or more silicon atoms, p and q are 0, 1, or 2, provided that p+q=1 or 2, and that when p+q=1, t=1.5, and when p+q=2, t=1; r is 0 or 1, provided that when r=0, t=1.5, and when r=1, t=1; m is an integer greater than or equal to 2; and n is a number from 0.01 to 100; B is represented by Formula IV below:
SiO2/(R4vSiOt)nxe2x80x83xe2x80x83(Formula IV)
where R4 is selected from the group consisting of hydroxyl, fluorine, alkoxy (e.g. methoxy), aryloxy, substituted siloxane, protein, peptide, carbohydrate, nucleic acid, and 1.5, and when v=2, t=1; and n is a number from 0.01 to 100; said interior of said particle having a composition of A, said exterior surface of said particle having a composition represented by A and B, and where said exterior composition is between about 1 and about 99% of the composition of B and the remainder including A. In the above particles, R4 may be represented by:
xe2x80x94OSi(R5)2xe2x80x94R6xe2x80x83xe2x80x83(Formula V)
where R5 is selected from a group consisting of a C1 to C6 straight, cyclic, or branched alkyl, aryl, or alkoxy group, a hydroxyl group, or a siloxane group, and R6 is selected from a group consisting of a C1 to C36 straight, cyclic, or branched alkyl (e.g. C18, cyanopropyl), aryl, or alkoxy group, where the said groups of R6 are unsubstituted or substituted with one or more moieties selected from the group consisting of halogen, cyano, amino, diol, nitro, ether, carbonyl, epoxide, sulfonyl, cation exchanger, anion exchanger, carbamate, amide, urea, peptide, protein, carbohydrate, and nucleic acid functionalities.
For attaching proteins or peptides to the surface of a silica particle, the particle may be treated with an aldehyde-containing silane reagent. MacBeath, et al. (2000) Science 289:1760-1763. Aldehydes react readily with primary amines on the proteins to form a Schiff base linkage. The aldehydes may further react with lysines. Alternatively, proteins, peptides, and other target molecules may be attached to the surface of the silica particle by using N-{m-{3-(trifluoromethyl)diazirin-3-yl}phenyl}-4-maleimidobutyramide which carries a maleimide function for thermochemical modification of cysteine thiols and an aryldiazirine function for light-dependent, carbene mediated binding to silica particles. Collioud, et al. (1993) Bioconjugate 4:528-536. Activation of a carbene-generating aryldiazirine with a 350-nm light source has been shown to lead to covalent coupling of proteins, enzymes, immunoreagents, carbohydrates, and nucleic acids under conditions such that biological activity is not impaired. Proteins or peptides can also be attached to the surface of a silica particle by derivatizing the surface silanol groups of the silica particle with 3-aminopropyl-triethoxysilane (APTS), 3-NH2(CH2)3Si(OCH2CH3)3. Han, et al. (1999) J. Am. Chem. Soc. 121:9897-9898.
In an example of binding a carbohydrate to the surface of a silica particle, an octagalactose derivative of calix{4}resorcarene is obtained by the reaction of lactonolactone with octaamine. Fujimoto, et al. (1997) J. Am. Chem. Soc. 119:6676-6677. When a silica particle is dipped into an aqueous solution of the octagalactose derivative, the resulting octagalactose derivative is readily adsorbed on the surface of the silica particle. The interaction between the octagalactose derivative and the silica particle involves hydrogen bonds. Ho Chang, et al., U.S. Pat. No. 4,029,583 describes the use of a silane coupling agent that is an organosilane with a silicon functional group capable of bonding to a silica particle and an organic functional group capable of bonding to a carbohydrate moiety.
For bonding oligonucleotides to the surface of a silica particle, the silica particle may be treated with xcex3-aminopropyl-triethoxysilane (APTES) to generate aminosilane-modified particles. The aminosilane-modified particles were then treated with p-nitrophenylchloroformate (NPC) (Fluka), glutaraldehyde (GA) (Sigma), maleic anhydride (MA) (Aldrich) and then treated with 5xe2x80x2-NH2-labeled DNA or 5xe2x80x2-SH-labeled DNA. Yang, et al. (1998) Chemistry Letters, pp. 257-258. Alternatively, oligonucleotides can be added to the surface of a silica particle by reacting 3-glycoiodoxypropyltrimethoxysilane with a silica particle bearing silanol groups and then cleaving the resulting epoxide with a diol or water under acidic conditions. Maskos, et al. (1992) Nucleic Acids Research 20(7): 1679-1684. Oligonucleotides can also bind to the surface of a silica particle via a phosphoramidate linkage to a silica particle containing amine functionalities. For example, silica particle containing an amine functionality was reacted with a 5xe2x80x2-phorimidazolide derivative. Ghosh, et al. (1987) Nucleic Acids Research 15(13):5353-5373. A 5xe2x80x2-phosphorylated oligonucleotide was reacted with the amine groups in the presence of water soluble 1-ethyl-3-(3-dimethylaminopropyl)-carbodimide (EDC) in N-methylimidazole buffer. Light directed chemical synthesis can be used to attach oligonucleotides to the surface of a silica particle. To begin the process, linkers modified with photochemically removable protecting groups are attached to a solid substrate. Light is directed through a photolithographic mask to specific areas of the surface, activating those areas for chemical coupling. Lipshutz, et al. (1993) BioTechniques 19(3):442-447.
In an embodiment, R6 may be greater than about 2.5 xcexcmol/m2, more preferably greater than about 3.0 xcexcmol/m2, and still more preferably greater than about 3.5 xcexcmol/m2. In a preferred embodiment, the surface concentration of R6 is between about 2.5 and about 3.7 xcexcmol/m2.
This invention also provides a bulk material including a population of the above particles where the particles have a mean particle size of about 0.5 to 100 xcexcm, more preferably a mean particle size of about 1 to 20 xcexcm. In an example, the particles may have a specific surface area of about 50 to 800 m2/g, more preferably about 100 to 200 m2/g. In an embodiment, the particles have specific pore volumes of about 0.25 to 1.5 cm3/g, more preferably about 0.5 to 1.0 cm3/g. In an example, the particles of the invention may have an average pore diameter of about 50 to 500 xc3x85, more preferably about 100 to 300 xc3x85.
Within the scope of the invention are separation devices (e.g., chromatographic columns, filtration membranes, sample clean up devices, and microtiter plates) including the above particles. For example, a chromatographic separation may be performed by running a sample through a column containing particles of the invention.
A method of preparing chromatographic particles for performing separations or for participating in chemical reactions, including: (a) prepolymerizing a mixture of an organoalkoxysilane and a tetraalkoxysilane (e.g., tetramethoxysilane and tetraethoxysilane) in the presence of an acid catalyst to produce a polyalkoxysiloxane; (b) preparing an aqueous suspension of said polyalkoxy siloxane, said suspension further comprising a surfactant, and gelling in the presence of a base catalyst so as to produce porous particles having silicon C1 to C7 alkyl groups, substituted or unsubstituted aryl groups, substituted or unsubstituted C1 to C7 alkylene, alkenylene, alkynylene, or arylene groups; (c) modifying the pore structure of said porous particles by hydrothermal treatment; and (d) replacing one or more surface C1 to C7 alkyl groups, substituted or unsubstituted aryl groups, substituted or unsubstituted C1 to C7 alkylene, alkenylene, alkynylene, or arylene groups of the particle with hydroxyl, fluorine, alkoxy, aryloxy, or substituted siloxane groups.
In the above method, the replacing may involve reacting the hybrid particle with aqueous H2O2, KF, and KHCO3 in an organic solution. In an embodiment, the molar ratio of organotrialkoxysilane to tetraalkoxysilane is about 100:1 to 0.01:1. Alkylphenoxypolyethoxyethanol may be used as surfactant in the above method. The above suspension may further include a porogen.
The porous inorganic/organic hybrid particles of the invention may have a surface concentration of silicon-methyl groups that is less than about 0.5 xcexcmol/m2, and a concentration of internal silicon-methyl groups such that over 10% of the internal silicons are silicon-methyl.
The porous inorganic/organic hybrid particles of the invention may have a surface concentration of the bonded phase alkyl groups that is greater than about 2.5 xcexcmol/m2, and a concentration of internal silicon-methyl groups such that over 10% of the internal silicons are silicon-methyl.
The surface concentration of silicon-methyl groups may be less than about 0.5 xcexcmol/m2, preferably between about 0.1 and about 0.5 xcexcmol/m2, more preferably between about 0.25 and about 0.5 xcexcmol/m2. The hybrid material may have a surface concentration of silanol groups greater than about 5.5 xcexcmol/m2, more preferably between about 5.5 and 6.8 xcexcmol/m2. The surface concentration of the bonded phase alkyl groups is generally greater than about 3.0 xcexcmol/m2, more preferably greater than about 3.5 xcexcmol/m2, still more preferably between about 2.5 and about 3.7 xcexcmol/m2. In an embodiment, the hybrid material has a concentration of internal silicon-methyl groups such that over 25% of the internal silicons are silicon-methyl
The hybrid material may have a bonded phase such as C18, C8, cyanopropyl, or 3-cyanopropyl.
In an embodiment, the hybrid particles have an average pore diameter of between about 130 and about 200 xc3x85, more preferably between about 160 and about 200 xc3x85. The average particle size is generally between about 5 and 6 xcexcm, more preferably about 5.4 to about 5.9 xcexcm.
The above hybrid materials have increased stability at low pH (e.g., below 4, below 3, below 2). In a method of performing high performance liquid chromatography a sample at a pH below 3, below 4, or below 5 may be run through a column containing one of the above hybrid materials.
In another alternative embodiment, this invention pertains to a method of forming a porous inorganic/organic hybrid material comprising: (a) forming a porous inorganic/organic hybrid particle having surface silicon-methyl groups; (b) replacing one or more surface silicon-methyl groups of the hybrid particle with hydroxyl groups (e.g. by reacting the hybrid particle with H2O2, KF, and KHCO3 in an organic solution); (c) bonding one or more alkyl groups to the surface of the porous inorganic/organic hybrid particle; (d) replacing one or more surface silicon-methyl groups with fluorine groups (e.g. by reacting the hybrid particle with H2O2, KF, and KHCO3 in an organic solution); and (e) capping the surface of the hybrid particle with trimethylchlorosilane.
Porous inorganic/organic hybrid particles may be made as described below and in the specific instances illustrated in the Examples. Porous spherical particles of hybrid silica may, in a preferred embodiment, be prepared by a four-step process. In the first step, an organotrialkoxysilane such as methyltriethoxysilane, and a tetraalkoxysilane such as tetraethoxysilane (TEOS) are prepolymerized to form polyalkylalkoxysiloxane (PAS) by co-hydrolyzing a mixture of the two components in the presence of an acid catalyst. In the second step, the PAS is suspended in an aqueous medium in the presence of a surfactant and gelled into porous spherical particles of hybrid silica using a base catalyst. In the third step, the pore structure of the hybrid silica particles is modified by hydrothermal treatment, producing an intermediate hybrid silica product which may be used for particular purposes itself, or desirably may be further processed below. The above three steps of the process allow much better control of the particle morphology, pore volume and pore sizes than those described in the prior art, and thus provide the chromatographically-enhancing pore geometry.
In the fourth step, the remaining surface silanol groups of the hybrid silica are derivatized into organic functional groups, such as by reacting with a halopolyorganosilane such as octadecyldimethylchlorosilane. The surface of the thus-prepared material is then covered by the alkyl groups embedded during the gelation and the organic groups added during the derivatization process. The surface coverage by the overall organic groups is higher than in conventional silica-based packing materials, and subsequently the surface concentration of the remaining silanol groups in the hybrid silica is smaller. The resulting material, used as a stationary phase for LC, shows excellent peak shape for base analytes, and better hydrolytic stability than other silica-based packing materials.
Where the prepolymerization step involves co-hydrolyzing a mixture of the two components in the presence of an acid catalyst, the content of the organotrialkoxysilane can be varied, e.g., from 0.2 to 0.5 mole organotrialkoxysilane per mole of tetraalkoxysilane. The amount of the water used for the hydrolysis can be varied, e.g., from 1.10 to 1.35 mole water per mole of the silane. The silane, water and the ethanol mixture, in the form of a homogeneous solution, is stirred and heated to reflux under a flow of argon. After refluxing for a time sufficient to prepolymerize and form polyalkylalkoxysiloxane, the solvent and the side product, mainly ethanol, is distilled off from the reaction mixture. Thereafter, the residue is heated at an elevated temperature, e.g., in the range of 120 to 140xc2x0 C. under an atmosphere of argon for a period of time, e.g., 1.5 to 16 h. The residue is further heated at this temperature, e.g., for 1 to 3 h under reduced pressure, e.g., 10xe2x88x922-10xe2x88x923 torr, to remove any volatile species.
In the second step, the PAS is suspended into fine beads in a solution containing water and ethanol at 55xc2x0 C. by agitation. The volume percent of ethanol in the solution is varied from 10 to 20%. A non-ionic surfactant such as TRITON X-100 or TRITON X-45 is added into the suspension as the suspending agent. The surfactant, having a structure of alkylphenoxypolyethoxyethanol, is believed to be able to orient at the hydrophobic/hydrophilic interface between the PAS beads and the aqueous phase to stabilize the PAS beads. The surfactant is also believed to enhance the concentration of water and the base catalyst on the surface of the PAS beads during the gelation step, through its hydrophilic groups, which induces the gelling of the PAS beads from the surface towards the center. Use of the surfactant to modulate the surface structure of the PAS beads stabilizes the shape of the PAS beads throughout the gelling process, and minimizes or suppresses formation of particles having xe2x80x9cshell-shapedxe2x80x9d morphology. A xe2x80x9cshell-shapedxe2x80x9d morphology is undesirable because it reduces mass transfer rates, leading to lower efficiencies.
The gelation step is initiated by adding the basic catalyst, e.g., ammonium hydroxide into the PAS suspension agitated at 55xc2x0 C. Thereafter, the reaction mixture is agitated at the same temperature to drive the reaction to completion. Ammonium hydroxide is preferred because bases such as sodium hydroxide are a source of unwanted cations, and ammonium hydroxide is easier to remove in the washing step. The thus-prepared hybrid silica is filtered and washed with water and methanol free of ammonium ions, then dried.
In an embodiment, the pore structure of the as-prepared hybrid material is modified by hydrothermal treatment, which enlarges the openings of the pores as well as the pore diameters, as confirmed by BET nitrogen (N2) sorption analysis. The hydrothermal treatment is performed by preparing a slurry containing the as-prepared hybrid material and a solution of organic base in water, heating the slurry in an autoclave at an elevated temperature, e.g., about 143 to 168xc2x0 C., for a period of about 6 to 28 h. The pH of the slurry is adjusted to be in the range of about 8.0 to 9.0 using concentrated acetic acid. The concentration of the slurry is in the range of 1 g hybrid material per 4 to 10 ml of the base solution. The thus-treated hybrid material is filtered, and washed with water and acetone until the pH of the filtrate reaches 7, then dried at 100xc2x0 C. under reduced pressure for 16 h. The resultant hybrid materials show average pore diameters in the range of about 100-300 xc3x85.
The surface of hybrid silica prepared so far still contains silanol groups, which can be derivatized by reacting with a reactive organosilane. The surface derivatization of the hybrid silica is conducted according to standard methods, for example by reaction with octadecyldimethylchlorosilane in an organic solvent under reflux conditions. An organic solvent such as toluene is typically used for this reaction. An organic base such as pyridine or imidazole is added to the reaction mixture to catalyze the reaction. The thus-obtained product is then washed with water, toluene and acetone and dried at 100xc2x0 C. under reduced pressure for 16 h. The resultant hybrid silica can be further reacted with a short-chain silane such as trimethylchlorosilane to endcap the remaining silanol groups, by using a similar procedure described above.
The surface of the hybrid silica particles may also be surface modified with a surface modifier, e.g., Za(Rxe2x80x2)bSixe2x80x94R, where Z=Cl, Br, I, C1-C5 alkoxy, dialkylamino, e.g., dimethylamino or trifluoromethanesulfonate; a and b are each an integer from 0 to 3 provided that a+b=3; Rxe2x80x2 is a C1-C6 straight, cyclic or branched alkyl group, and R is a functionalizing group, and by polymer coating. Rxe2x80x2 may be, e.g., methyl, ethyl, propyl, isopropyl, butyl, t-butyl, sec-butyl, pentyl, isopentyl, hexyl or cyclohexyl; preferably, Rxe2x80x2 is methyl.
The functionalizing group R may include alkyl, aryl, cyano, amino, diol, nitro, cation or anion exchange groups, or embedded polar functionalities. Examples of suitable R functionalizing groups include C1-C20 alkyl such as octyl (C8) and octadecyl (C18); alkaryl, e.g., C1-C4-phenyl; cyanoalkyl groups, e.g., cyanopropyl; diol groups, e.g., propyldiol; amino groups, e.g., aminopropyl; and embedded polar functionalities, e.g., carbamate functionalities such as disclosed in U.S. Pat. No. 5,374,755 and as detailed hereinabove. In a preferred embodiment, the surface modifier may be a haloorganosilane, such as octyldimethylchlorosilane or octadecyldimethylchlorosilane. Advantageously, R is octyl or octadecyl.
Polymer coatings are known in the literature and may be provided generally by polymerization or polycondensation of physisorbed monomers onto the surface without chemical bonding of the polymer layer to the support (type I), polymerization or polycondensation of physisorbed monomers onto the surface with chemical bonding of the polymer layer to the support (type II), immobilization of physisorbed prepolymers to the support (type III), and chemisorption of presynthesized polymers onto the surface of the support (type IV). see, e.g., Hanson et al., J. Chromat. A656 (1993) 369-380.
The current state of the art hybrid organic/inorganic based RP HPLC column packing is prepared by bonding chlorosilanes to a hybrid particle. The hybrid particle has a methyl-silicon group incorporated throughout the particle""s structure, that is, the methyl group is found in both the internal framework of the hybrid silicate backbone as well as on the particle""s external surface. Both the internal and external methyl groups have been shown to contribute to the hybrid""s improved stability in high pH mobile phases when compared to purely silica based materials. However, the surface methyl groups also lead to lower bonded phase surface concentrations after bonding with silanes, e.g., C18 and C8 silanes, in comparison to silica phases, presumably because the methyl groups on the surface are unreactive to bonding. For example, when using low pH (e.g., about pH 5) mobile phases, a hybrid product such as XTerra(trademark) MS C18, which has a trifunctional C18 bonded phase, is less stable compared to conventional silica based trifunctional C18 bonded phases. The surface methyl groups of the hybrid particle may decrease the level of cross-bonding between adjacent C18 ligands, essentially the methyl groups block the connection. This effect would be expected to reduce low pH stability, since the C18 ligand has fewer covalent bonds to the surface.
The present invention provides a procedure to selectively convert surface silicon-methyl groups with silanol groups. Depending on the reaction conditions, the particle""s internal framework is not disturbed or is only slightly disturbed leaving the internal methyl groups unaffected. This then results in a particle different from the original hybrid particle, where the surface now more resembles that of a pure silica particle. The particle""s new composition is supported by standard analytical analysis (CHN, BET, NMR) as well as the finding that a neutral analyte, acenaphthene, is less retained under reversed-phase conditions in comparison to the unmodified hybrid particle. Presently, the modified particle is also found to be less stable under basic pH conditions, a result due to the surface methyl groups no longer being present to protect the surface. At the same time, these modified particles have been found to afford a high C18 surface concentration after bonding with chlorosilanes, arguably due to the newly formed surface silanols being converted to ligand siloxanes. These bonded particles were found to give a 2.7 fold increase in low pH stability. The result is attributed to the high surface concentration of C18 ligand, which then permits a higher degree of cross-bonding between adjacent C18 ligands and hence more covalent bonds between the ligand and particle surface. Consistent with this model, peak tailing for basic analytes increased, and high pH stability decreased for the modified C18 phase versus the standard hybrid C18 bonded phase. Both can be attributed to the increased silanol population in the modified particle""s surface.
Sixe2x80x94CH3 groups at the surface of the hybrid particle can be converted into Sixe2x80x94OH and Sixe2x80x94F groups by the following reaction 
The above reaction is run in methanol/THF/water, so full wetting and total pore access should be possible. The mechanism of cleavage appears to be a modified Baeyer-Villager oxidation, which should have a minimal transition state requirement. Methyl loss may be measured by e.g. CHN combustion analysis of the reacted product, where the reduction in %C of reacted versus untreated is taken as a measure of surface methyl groups lost and hence present on the surface. IR and NMR analysis could also be used to measure this change as well as look for any other surface changes.
Other fluorinating reagents can be used in place of KF. For example, potassium hydrogen fluoride (KHF2), tetrabutylammonium fluoride ({CH3CH2CH2CH2}4NF), boron trifluoride-acetic acid complex (BF3-2{CH3CO2H}), or boron hydrogen tetrafluoride diethyl etherate (HBF4-O(CH2CH3)2) can be used in place of KF.
Other carbonate reagents, such as sodium hydrogencarbonate, for example, can be used in place of potassium hydrogencarbonate.
Other reagents can be used in place of hydrogen peroxide (H2O2). For example, 3-chloroperoxybenzoic acid (ClC6H4CO3H) and peracetic acid (CH3CO3H) can be used in place of hydrogen peroxide (H2O2).
Alternatively, silicon-carbon bonds can be cleaved by reacting the silicon compound with m-chloroperbenzoic acid (MCPBA) as shown below. A description of this synthesis can be found in Tamao, et al. (1982) Tetrahedron 39(6):983-990.
Similarly, silicon-carbon bonds can be cleaved by reacting the silicon compound with hydrogen peroxide as shown below. A description of this synthesis can be found in Tamao, et al. (1983) Organometallics 2:1694-1696. 
The porous inorganic/organic hybrid particles have a wide variety of end uses in the separation sciences, such as packing materials for chromatographic columns (wherein such columns will have extended lives), thin layer chromatographic (TLC) plates, filtration membranes, microtiter plates, and the like having a stationary phase which includes porous inorganic/organic hybrid particles having a chromatographically-enhancing pore geometry. The stationary phase may be introduced by packing, coating, impregnation, etc., depending on the requirements of the particular device. In a particularly advantageous embodiment, the chromatographic device is a packed chromatographic column, e.g., HPLC.
The present invention may be further illustrated by the following non-limiting examples describing the preparation of porous inorganic/organic hybrid particles, and their use.